Effects of therapeutic hypothermia on myocardial Ca2+/calmodulin-dependent protein kinase II and autophagy after cardiopulmonary resuscitation in swine

Abstract

Objective To investigate the effects of therapeutic hypothermia (TH) on myocardial Ca2+/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) and cell autophagy after cardiopulmonary resuscitation (CPR) in swine. Methods Twenty healthy male domestic swine weighing 33-40 kg were randomly (random number) divided into 3 groups: sham group (n=4), CPR group (n=8) and TH group (n=8). Sham animals only underwent general preparation without experiencing cardiac arrest and resuscitation. The animal model was established by 8 min of electrically induced ventricular fibrillation and then 5 min CPR in the CPR and TH groups. Successful resuscitation was regarded as an organized rhythm with a mean arterial pressure of greater than 50 mmHg for 5 min or more. After successful resuscitation, body temperature was decreased to 33 ℃ by a cooling blanket and then maintained until 24 h post-resuscitation, and followed by a rewarming at a rate of 1 ℃/h for 5 h in the TH group. A normal temperature was maintained by the blanket throughout the experiment in the sham and CPR groups. At 6, 12, 24 and 30 h after resuscitation, the values of stroke volume (SV) and global ejection fraction (GEF) were measured by PiCCO, and meanwhile the serum concentrations of cardiac troponin I (cTnI) were measured by ELISA assay and the serum activities of creatine kinase-MB (CK-MB) were evaluated by an automatic biochemical analyzer. At 30 h after resuscitation, the animals were sacrificed and left ventricular myocardium was obtained for the determination of CaMKⅡ, microtubule-associated protein light chain 3Ⅱ (LC3Ⅱ) and p62 expressions by Western blot. The variables were compared with One way analysis of variance and then the Bonferroni test among the three groups. Results Compared with the sham group, myocardial dysfunction and injury after resuscitation were observed in the CPR and TH groups, which were indicated by decreased SV and GEF and also increased cTnI concentration and CK-MB activity in serum (all P<0.05). Compared with the CPR group, the values of SV and GEF were significantly increased at 6 h after resuscitation, and serum cTnI concentration and CK-MB activity were significantly decreased starting 12 h after resuscitation in the TH group [SV (mL): 25.0±6.9 vs 31.9±3.3 at 6 h, 26.7±5.1 vs 34.6±3.7 at 12 h, 28.8±3.3 vs 35.7±3.2 at 24 h, 29.2±5.2 vs 36.7±3.3 at 30 h; GEF (%): 17.1±2.7 vs 19.9±1.8 at 6 h, 18.7±1.9 vs 21.6±1.8 at 12 h, 19.3±2.3 vs 23.0±2.4 at 24 h, 21.0±1.7 vs 23.7±1.7 at 30 h; cTnI (pg/mL): 564±51 vs 466±56 at 12 h, 534±38 vs 427±60 at 24 h, 476±55 vs 375±46 at 30 h; CK-MB (U/L): 803±164 vs 652±76 at 12 h, 693±96 vs 557±54 at 24 h, 633±91 vs 480±77 at 30 h, all P<0.05]. Tissue detection indicated that the expression of CaMKⅡ and LC3Ⅱ were increased while the expression of p62 was decreased in post-resuscitation myocardium in the CPR and TH groups compared with the sham group (all P<0.05). However, the expression of CaMKⅡ and LC3Ⅱ were decreased and the expression of p62 was increased in post-resuscitation myocardium in the TH group compared to the CPR group (CaMKⅡ: 0.73±0.06 vs 0.58±0.05; LC3Ⅱ: 0.69±0.09 vs 0.50±0.07; p62: 0.40±0.07 vs 0.68±0.14, all P<0.05). Conclusion The mechanism of TH alleviating post-resuscitation myocardial dysfunction and injury may be related to the inhibition of CaMKⅡ expression and cell autophagy. Key words: Cardiac arrest; Cardiopulmonary resuscitation; Myocardial dysfunction; Cardiac injury; Therapeutic hypothermia; Ca2+/calmodulin-dependent protein kinaseⅡ; Autophagy; Swine